Referring to FIG. 1 (PRIOR ART), there is a block diagram illustrating the basic components of an exemplary fluorescence array imager 100 which is widely used today in the fields of chemistry, biochemistry and molecular biology. The exemplary fluorescence array imager 100 shown includes an excitation source 102 (e.g., laser, lamp, light emitting diode) which emits an optical beam 104 that is reflected by a dichroic mirror 106 and passed through a lens 108 before it illuminates a test sample 110 (which contains fluorescently labeled materials). In this example, the test sample 110 is a protein array 110 that is located within a well of a microplate 112.
The fluorescence array imager 100 also includes an imaging device 114 (e.g., charge-coupled device (CCD), photomultiplier tube (PMT)) which captures (receives) the fluorescence 105 that is emitted from the protein array 110. More specifically, the imaging device 114 captures the emitted fluorescence 105 after it passes through the lens 108, the dichroic mirror 106 and a filter 116. Then, the imaging device 114 creates and outputs a two-dimensional (2D) fluorescence image of the protein array 110. This is all possible, because when photons of a certain wavelength are emitted from the excitation source 102 and absorbed by certain molecules in the protein array 110, then those molecules emit fluorescence 105 at another wavelength. And, this emitted fluorescence 105 is what is captured by the imaging device 114.
In particular, the fluorescence array imager 100 implements one of two methods to create the 2D fluorescence image: (1) a narrow beam of light 104 (emitted from a laser 102) can be scanned across a region of interest on the protein array 110 and the emitted fluorescence 105 from each scanned location is collected by the PMT 114 and then later digitally composed into the 2D fluorescence image; or (2) a wide beam of light 104 (emitted from a lamp 102) can be used to illuminate the whole region of interest on the protein array 110 and the emitted fluorescence 105 from this region is collected by the CCD 114 (or a 2D photo detector 114) and directly composed into the 2D fluorescence image. In either case, the resulting 2D fluorescence image represents the intensity of the fluorescence 105 emitted from the molecules within the protein array 110.
A principle assumption in fluorescence imaging when a quantitative analysis is used to determine the concentration of molecules within a protein array 110 is that the intensity of the excitation source 102 is locally uniform, and the collection optics 106, 108 and 116 and imaging device 114 are uniform, and therefore the intensity of the emitted fluorescence 105 is going to have a linear relationship to the concentration of the molecules of interest. In view of this assumption, it follows that the intensity of the emitted fluorescence within the 2D fluorescence image can with a simple calibration be directly converted to indicate the concentration of the molecules of interest. Unfortunately, the assumption that the intensity of the excitation source 102 is locally uniform is not necessarily a correct assumption.
In PMT-based scanning fluorescence imaging techniques, the most commonly used excitation light source 102 is a laser because it can be focused to a very small optical beam 104 which then scans the region of interest on the protein array 110. The excitation energy of the scanned region of interest is going to be uniform if the laser's output intensity is stable in the time while the protein array 100 is scanned. However, the laser's output intensity often fluctuates and decays as a function of time which can cause artificial errors to be introduced into the 2D fluorescence image. This is not desirable.
In CCD-based fluorescence imaging techniques, the most commonly used excitation light source 102 is a lamp because it can better illuminate a sizable region of the protein array 110 when compared to a laser. However, the intensity of the lamp 102 is not always uniform which can cause artificial errors to be introduced into the 2D fluorescence image. In addition, the CCD 114 and collection optics may not be perfectly uniform because of: (1) the aging of the CCD 114; and (2) the accumulation of dust and dirt over time on the CCD 114 and collection optics. This is not desirable.
Lasers have also been used as an illumination source in CCD-based fluorescence imaging, however, the Gaussian distribution of a laser beam does not allow a quantitative measurement without a calibration of the laser's intensity. In the past, top-hat optics has been used in an attempt to help generate a relative uniform distribution of laser intensity. However, the use of top-hat optics sacrifices the total intensity output of the laser which would then limit the sensitivity of the measurement. This is not desirable.
Accordingly, there has been a need to address these shortcomings so one can use a fluorescence array imager 100 to obtain an “accurate” 2D fluorescence image of a protein array 110 (or other analyte array 110). In particular, there has been a need to obtain an “accurate” 2D fluorescence image which represents the concentration of the molecules of interest within the protein array 110 (or other analyte array 110) without being adversely affected by a non-uniform excitation source 102. This need and other needs are addressed by the present invention.